The ubiquitin system is the major pathway for the controlled degradation of intracellular proteins in eukaryotic cells. The ubiquitin system regulates the half-life of cellular proteins including, cyclins, cyclin dependent kinase inhibitors, histones, oncoproteins and tumor suppressors and is an important regulatory component of cell cycle progression, endocytosis, receptor regulation, transcription, signal transduction, oncogenesis, apoptosis and antigen presentation (Hershko et al., J. Biol. Chem., 269:4940-46). Abnormalities in the ubiquitin pathway are implicated in many pathological conditions including cancer (Johnston, 1999).
In the ubiquination pathway targeted proteins are marked with a chain of four or more ubiquitins that are covalently attached through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lys1 residue in the substrate protein. The chain of ubiquitins mark the protein for degradation by a multi-subunit ATP-dependent protease known as the 26S proteasome enzyme complex. The degradation process is catalyzed in a sequential reaction involving three enzymes: E1 a ubiquitin activating enzyme, E2 an ubiquitin-conjugating enzyme, and E3 an ubiquitin-protein ligase enzyme. E1, in an ATP dependent process, forms a thiol ester bond with the C-terminus of ubiquitin. The activated ubiquitin is then transferred to a catalytic cysteine on one of several E2 enzymes. The E2 then donates the ubiquitin directly to the protein substrate or by association with an E3 protein (ubiquitin ligase; in most cases a polyprotein complex). After ligation of the first ubiquitin to the substrate protein, further ubiquitins are usually targeted to the first ubiquitin to form multi-ubiquitin chains. The 26S proteasome then catalyzes the degradation of the ubiquitin tagged proteins. Disruptions in the regulation of protein degradation by the ubiquitin pathway can significantly impact pathways in which correct protein concentration and half-life are essential, e.g., cell cycle.
Thus, defects in the ubiquitin pathway are likely to cause excessive proliferation and transformation in cells and thus inhibitors of this pathway are needed. However, because the ubiquitin pathway plays an essential role in many biological pathways, non-selective compounds are likely to be toxic. Thus, targeting specific ubiquitin enzymes may overcome the obstacle of toxicity. An attractive target in this pathway are the E2, ubiquitin conjugating enzymes. However, there are currently no compounds that selectively target E2. Thus there is a need in the art to develop safe and effective compounds that selectively inhibit ubiquitin conjugating enzymes or E2s.